Liquidborne Particle Counting using Light Obscuration and Light

Scattering Methods

2

What has been . . .

Focus has been on injectable liquids

• Possibility to block capillaries and arteries– Red Blood cells are about 5 µm

– Capillary (5 to 10 µm)

– Large veins (10 to 50 µm)

• Threat of microbial infection

• Allergic reaction to foreign substances

4

Definition of Particulate Contaminants

Unwanted insoluble matter that exist as “randomly-sourced extraneous substances”

• Excludes homogeneous monotonic materials that exist as a precipitate or suspension

• i.e. colloids, drug degradation or otherwise derived from a defined source and can be analyzed by chemical means

Regarded as “contamination” and “adulteration” under Food and Drug Act

• the chemical composition of the particulate is varied, and

would not be declared on the label– Examples: bits of paper fiber, fragments of filler material, etc

5

Liquid Particle Counting Applications

Final Product Testing – USP <788>

• SVP or SVI (Small Volume Parenteral/Injectable)– Ampoules, Vials

• LVP or LVI (Large Volume Parenteral/Injectable)– IV (Intravenous) solutions

Process contamination studies

Decomposition studies (stability)

DI or WFI Water

Precision Cleaning – Medical Devices

• Aqueous

• Other Chemicals

6

Other Applications for Particle Counting

Medical Devices

• Cleanliness of manufacturing environment

• Cleanliness of device before implantation– pacemakers, stents, artificial arteries

• Cleanliness of reclaimed devices

Design of particulate-based medicines

• Inhalation therapies

• Intentional occlusion of blood flow to cancers

• Time-based dosages

• Transdermal absorption

7

Global Regulations: Particles in Liquids

USP <788>, EP 2.9.19, JP XV, KP, CP

Primary method

• Optical Particle Counter [OPC]– Light Obscuration Counter

Secondary method

• Optical microscope– Subjective

– Labor intensive

– Requires more time to process samples

8

Proposed: USP 787, USP 1787

USP <787>

Under discussion

Focused on reducing necessary test volumes due to concerns of biotechnology manufacturers of cost for test

Expensive and often very small dose factory

– for example: 500 uL pre-filled syringe

9

Proposed: USP 787, USP 1787

USP <787>

Primary method ?

• Optical Particle Counter [OPC]– Light Obscuration Counter

Secondary method ?

• Optical microscope– Subjective

– Labor intensive

– Requires more time to process samples

10

Proposed: USP 787, USP 1787

USP <787>

Small sample volume

- 1 mL ??

Concerns with variability

- within production lots

- in analytical methods

11

Optical Particle Counter

Optical Instrument

• Must move fluid through sensor

• Can quantify particles from 100 nm to 5000 µm

• Counts particles individually (one at a time)

• Cannot tell you composition

• But results are immediate

12

Alumino-silicate with K and Ti

Talc

Many shapes and sizes

13

Sizing Particles by Microscope

Largest Dimension

d

Ferret’s Diameter

d

Projected Area

d

Martin’s Diameter

Area A

Area B

d

14

Challenges of Protein-based Products

Handling can change material !!!

• Agitation

• Heat and Light

• Contaminates

• Container: Vials versus syringes/cartridges

• Shear forces

Key concern is Aggregation

• Reduction of native form (impacts efficacy)

• Introduction of homogeneous aggregates

• Introduction of heterogeneous aggregates

15

Challenges of Protein-based Products

Transparency of most proteineous entities• Refractive index• NIST working on calibration material

Not “contamination” but instead a shift from native form• Not a solution as with small-molecule therapeutics• Formation of quaternary structures [dimers, etc.]• Protein complexes

Reconstitution of lyophilized product

16

Refractive Index

Key is the ability to distinguish between the particle and the surrounding fluid

- needs to be great enough

Optical response is proportional to comparative index

17

Refractive Index

Key is the ability to distinguish between the particle and the surrounding fluid

- needs to be great enough

Optical response is proportional to comparative index

18

Refractive Index

NIST working on protein-like calibration material

• Probably 2 years away

• Exploring 2 methods of manufacture

• Need thread-like material

• Indices near water

• Stable over reasonable period

19

II. Sample Handling

20

Settling/Agitation

Entrained gas

- sonication probably not ideal with protein structures

- light vacuum seems to work OK

Settling

Limits collection of particles

- especially of greater mass

- dependent on time and viscosity

- improved collection with slanted containers

21

Consistency of sample characteristics

Temperature

Settling

Probe position

22

Sampling Errors Account for most problems Accidental Contamination or Alteration by Technician

Issues with Sampling Particles in Liquids

1. System Preparation

Initial Cleanliness

Calibration

3. Sample Handling

Aggregation

Settling

Cavitation

2. Sample Preparation

Contamination - Particles

- Gases

- Liquids

24

Sizing Particles by Microscope

Largest Dimension

d

Ferret’s Diameter

d

Projected Area

d

Martin’s Diameter

Area A

Area B

d

25

HIAC Liquid Particle Counters

Example: HIAC 9703

• The industry standard liquid particle counter since 1997

• USP <788> was written specifically around HIAC technology

• Every major manufacturer of particle calibration standards uses the HIAC 9703

26

HIAC Liquid Particle Counters

Example: HIAC 9703+• Improved sample mounting

method for small vials or containers

• Detection of usual conditions such as bubbles or contamination

• Proven syringe sampler

• SVI and LVI sampling

• Addresses non-compendial applications, e.g. R&D and other low frequency, small sample volume applications

ReproducibilityRepeatability

27

Detection Ranges

1 10 100 1000 10000 100000 1000000

Light Scattering

Light Obscuration

1µm0.1µm 10µm 100µm

nm

28

Light Obscuration

Light Obscuration Sensors and system

• also known as Light Extinction

• also known as Light Blocking

29

Principles: Light Obscuration

Detector Output

30

Detector Output

Principles: Light Obscuration

31

Principles: Light Obscuration

32

Particle Detection

Like an air particle counter, the larger the particle, the larger the pulse that is created

33

Principles: Light Scattering

Laser Diode

Detector

Mirror

Light Trap

Detector Output

34

Laser Diode

Detector

Mirror

Particle

Light Trap

Principles: Light Scattering

35

Advantages: Light Scattering

Good sensitivity from 0,1µm to 50µm

Wide range of sample concentration

Good rejection of false counts

High sample flow rates

36

Disadvantages: Light Scattering

More complicated construction = higher cost

Characteristics of particle surface (shiny, color) affect response

37

Effect of colors and surfaces on Light Scattering

Dark Light Shiny

38

Alumino-silicate with K and Ti

Talc

39

Sizing Particles by Microscope

Largest Dimension

d

Ferret’s Diameter

d

Projected Area

d

Martin’s Diameter

Area A

Area B

d

40

General Comments on Liquid Counting

Particle Counters Report Size – But measure an Optical Response

– Difference in reported size compared to microscope

Calibration Relates the Optical Signal to Size

– Difference between calibration material characteristics and “real world” particles

Projected Area

d

41

General Comments on Liquid Counting

Particle Counters Report Size

• But measure an Optical Response

• Differences in reported size compared to microscope

Calibration Relates the Optical Signal to Size

• Difference between calibration material characteristics and “real world” particles

42

LO results versus LS results

Light Obscuration [LO]

• Good immunity to variations of surface and morphology

• Very stable

• Limit of quantitation circa 1.2 – 1.3 microns

Light Scattering [LS]Results affected by surface characteristics and coloring

Good stability

Limit of quantitation sub-micron

Problem can occur in the attempt to correlate results of these two methods above 1 micron

44

System Preparation

2-step Verification - optional:• Run 2 test solutions

– Blank (“particle-free” water)– Aqueous solution containing known counts

at 10 µm or 15 µmIn the range of 1000 to 3000 counts per mL

• Frequency – based on risk analysis– Each morning– Shift change– Change of operator– Other interval

45

System Preparation

Check for bubbles in sample lines and syringe• Affects flow rate and calibration

Verify correct calibration curve installed• Different flow rates for same sensor• Change of syringe size• Some companies have multiple sensors

Verify calibration is current• Sensor resolution and response curve• IST tests conducted [USP, JP]

46

System Preparation

Instrument Standardization Tests [IST]• Five tests of system

– Volume accuracy– Flow rate accuracy– Calibration of sensor– Resolution– Count accuracy

• Required by USP and JP but not EP– USP <788> 31

“…at intervals of not more than six months.”

– JP <24>“…at least once a year.”

47

Sample Preparation

Removing residue of previous sample• If previous sample contained particles, may be good

plan to run a “particle-free” blank before the next sample

• Use liquid that is compatible with sample fluid

– An aqueous blank could trigger false counts in an oil-based sample by causing immiscible droplets

– Potential residue from previous sample can cause change of counts

Data from first run of series is often discarded

48

Sample Preparation

Contamination

• Particles

• Gases

• Liquids

49

Sample Preparation

True Particle Sources• Residue on glassware and equipment• Ambient air• Paper dust• Glass• Diluent• Residue from previous sample• Colloidal suspensions

False Particle Sources• RF signals or other electronic interference• Bubbles from entrained gases

50

Sample Preparation

Work in controlled environment

Use particle-free gloves

Let water flow for 200 mL or more after opening a valve / tap

• Opening / closing valve generates particles

Wipe the outside of containers before sampling

• Particles on surface of vials or ampoules

Open vials and ampoules away from beaker or flask

• Particles from activity can fall into open container

• Wash outside of containers to reduce potential particle source

51

Degassing sample

Three common methods• Allowing to stand in ambient air

Risk of large particles settling

• Sonification [ultrasound]80 to 120 watts [USP <788>]30 seconds [USP <788>]

• Vacuum Bell jar or dessicator 0.6 – 0.8 atmospheres for 2 to 10

minutes

52

Sample Preparation

Possibility of decreasing true particle counts

• Settling

• Lack of agitation

• Position of probe in sample vessel

• Remaining material from previous sample run

– Sample with lower counts

– Blank

53

Sample Handling

Aggregation

Settling

Entrained gases

54

Sample Handling

Aggregation

• Samples held over time or at extremes of temperature can develop aggregates of smaller particles

• Exposure to light can also trigger this reaction

• Sub-micron particles can thus add to particle counts above 10 µm

• Suggestion:– Profile counts under 10 µm [e.g. at 2 or 5 µm] in addition

to standard count sizes at 10 and 25 µm

55

Sample Handling

Settling

• Undercounting caused by– Gravitational settling

– Failure to suspend particulate matter adhering to walls or stopper of container

• Standards have recommended procedure for agitation

– Multiple inversions of original container before decanting

– Constant motion of liquid during sampling“Gently stir the contents of the container by hand-swirling or by

mechanical means…” USP <788>

56

Optical Particle Counter

Instrument & sensor

• Must move fluid through sensor

• Can quantify particles from 100 nm to 5000 µm

• Counts particles individually (one at a time)

• Cannot tell you composition

• But results are immediate

57

Questions?

58

Patent Pending

59

New Hardware: 9703+

Key features

Auto stop for sensor elevator arm

Small vial holding clamp

Sample probe with reduced dead volume

Back-flush and forward flush from front panel

Supports MC-05 sensor (0.5 micron sensitivity)

60

Software: PharmSpec 3

Key new features

Compendial test support continues

USP, EP, JP, KP looks same as previous PharmSpec versions

Uses same log on as for Windows

Improved Report format

Improved Error Detection and Display

61

Still the HIAC 9703 you know and trust – only better!

Syringes

• 1 ml, 10 ml, 25 ml

Flow rate settings

• 10 to 100 ml

Sensors

• MC-05 is added

Sampling Probes

• added shorter small-bore probe

Instrument size / shape

• 50%+ of instruments are placed in laminar flow cabinets.

• Smooth, curved surfaces create less turbulence for the air flow

62

Easier, Faster and Confident Sampling

Use less sample, save valuable time - protect your investment

• Small vial clamp ensures that sample does not spill during testing

• Probe needle safety switch prevents probe damage

• New small needle probe with industry’s smallest tare volume

63

Easier, Faster and Confident Sampling

Reduce uncertainty from data anomalies

• Bubble alarm notification

• Contamination alarm notification

• Advanced notification when service or calibration is due

• Invalid configuration notification

64

HIAC just got easier!

Less time needed for clean-up• Automated flushing and cleaning routines

• Push a button, walk away and return to a clean sensor

Export your data with ease

• Select one, several or all of your historical data records with our batch export utility

• Select PDF, Word, Excel, or text files

Save time with electronic signature

• Stricter interpretation of 21CFR Part 11 electronic signature process…. WITHOUT more manual inputs

• Remembers user Login ID

65

HIAC 9703+ Flexibility

Interchangeable sampling probes, syringes, and sensorsEnsure you have one instrument to manage all

applications

Now supports MC05 sub-micron sensor

Change configuration with no impact to instrument validation

Customized reportingCustomize the number of reviewers and approvers

for compendial test reports

Add company logo, user-defined descriptors

Customized test recipesProcedure Builder enables the development of

unique test recipes for your application

Enables testing to marketing license-specific applications

66

New Sampling Probe

3 probes available

• Tare

¼” ID =1.2 ml tare volume

1/16” ID = 0.172 ml tare volume

• New small / short probe

1/16” ID = 0.09 ml tare volume

Tests can be performed on 1 ml of product!

67

Small Vial Clamp

Small Vial Clamp Platform

• Can be retrofitted

• Can be removed

Ease of Use

• Use one hand to compress lever arms

• Use other hand to place sample in central location

• Decompress hand

• Clamp auto-centers and holds sample container

68

Docking Module

Docking Module

• Enables removal/disabling of the stir bar mechanism

• Enables field installation of small vial clamp outside of the laminar flow cabinet

–Avoid potential of re-qualification that can occur if instrument is moved

Future developments to expand 9703 applications

69

Sampling Safety Switch

Sampling safety switch

• Ensures the sampling probe does not crash (and bend or break) into the docking module

• Ensures the probe does crash into or tip the sample container

70

Liquidborne Particle Counting using Light Obscuration and Light

Scattering Methods